Device for capturing circulating cells

ABSTRACT

The present invention provides devices and methods for capturing rare cells. The devices and methods described herein can be used to facilitate the diagnosis and monitoring of metastatic cancers.

This invention was made with government support under Grant No.CA119347, awarded by the National Institutes of Health. The U.S.Government has certain rights in this invention.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional Application No.61/161,248, filed on Mar. 18, 2009 and 61/301,839, filed on Feb. 5,2010, the entire contents of which are incorporated herein by reference,and is a U.S. national stage application under 35 U.S.C. §371 ofPCT/US/2010/027816 filed Mar. 18, 2010, the entire contents of which areincorporated herein by reference.

BACKGROUND

1. Field of Invention

The present invention relates to devices and methods for capturing rarecells.

2. Background Information

Cancer is one of the leading causes of death in the developed world,resulting in over 500,000 deaths per year in the United States alone.Over one million people are diagnosed with cancer in the U.S. each year,and overall it is estimated that more than 1 in 3 people will developsome form of cancer during their lifetime.

Most cancer patients are not killed by their primary tumor. Instead,cancer patients succumb to metastases: the spread of malignant cellsfrom one part of the body to another. If a primary tumor is detectedearly enough, it can often be eliminated by surgery, radiation,chemotherapy or some combination of these treatments. In contrast,metastatic tumors are difficult to detect and treatment becomes morechallenging as metastases progresses. As such, there is a need todevelop methods for detecting early-stage cancer metastasis.

Cancer cells that break away from the primary tumor site are known ascirculating tumor cells (CTCs).¹ CTCs represent a potential alternativeto invasive biopsies as a source of tumor tissue for the detection,characterization, and monitoring of non-hematologic cancers.²⁻⁴ Over thepast decade, CTCs have become an emerging “biomarker” for detectingearly-stage cancer metastasis, predicting patient prognosis, as well asmonitoring disease progression and therapeutic outcomes of cancer.⁵However, isolation of CTCs have been technically challenging due to theextremely low abundance (a few to hundreds per mL) of CTCs among a highnumber of hematologic cells (10⁹ cells/mL) in the blood.^(4,6,7)

Previous approaches for enriching or sorting CTCs from peripheral bloodinclude flow cytometry, immunomagnetic beads, high-throughputoptical-imaging systems, and fibre optic array scanning.Immunomagnetic-bead purification of CTCs is currently the most widelyused technology in the clinical setting, and has successfully identifiedCTCs in patients with lung, prostate, colon, breast, and pancreaticcancer.^(3,4,8-10) However, this approach isolates small numbers of CTCs(4±24 (mean±s.d.) per ml in lung; 11±118 in breast; 10±33 in prostate;and 1±2 in both colorectal and pancreatic cancers)³ with very low purity(0.01-0.1%)¹⁰, and low yield (˜20-60% of patients)³. The level of“biological noise” associated with the low sensitivity, selectivity, andyield of immunomagnetic-bead technologies restricts their use in earlycancer detection and in monitoring patient response to treatment. Atpresent, immunomagnetic-bead technology is useful as a gross prognostictool, classifying patients into high- and low-risk categories.⁵

Microfluidic lab-on-a-chip devices provide unique opportunities for cellsorting and rare-cell detection. Microfluidic technology has beensuccessfully used for microfluidic flow cytometry, continuous size-basedseparation¹¹ and chromatographic separation¹²; however, these methodsare unable to process large sample volumes (e.g., milliliters of wholeblood)¹³. Microfluidic technology has also been used to capture CTCsfrom whole blood samples.^(8,9) However, existing CTC-capture systemsrequire complicated fluidic handling systems to introduce blood flowthrough the devices. Furthermore, these systems use microstructures,which are not optimal for cell capture, to isolate CTCs.

The surfaces of most tumor cells of epithelial origin (carcinomas) arecovered with nanoscaled microvilli of variable sizes andconfiguration.¹⁴ In benign epithelial cells of glandular origin, themicrovilli are polarized (i.e., confined to one aspect of the normalcell, usually that facing the lumen of a gland or organ) and are ofuniform and monotonous configuration. The microvilli of epithelialcancer cells cover the entire cell surface, vary in size and length, andsometimes form clumps of very long microvilli. In some tumors, notablycarcinomatous mesothelioma, tufts of long microvilli characterize themalignant cells. Furthermore, additional structures are present on thecell surface, which are also nanoscale in size, including lamellipodia,filopodia, and lipid-rail molecular groups. Some embodiments of thepresent invention proposes a new generation of cell capture devices thattakes advantage of the presence of these nanoscaled structures on thecell surface.

SUMMARY

Further objectives and advantages will become apparent from aconsideration of the description, drawings, and examples.

A device for capturing cells according to embodiments of the presentinvention has a substrate containing a nanostructured surface region.Attached to the nanostructured surface region is a plurality of bindingagents, which are capable of selectively capturing target cells in acell sample. The nanostructured surface region contains a plurality ofnanostructures. The nanostructures have a longitudinal dimension and alateral dimension, and in some embodiments, the longitudinal dimensionis at least ten times greater than the lateral dimension.

In some embodiments or the present invention, the device is amicrofluidic device. The microfluidic device has a substrate attached toa flow layer, forming a microfluidic channel. The substrate has ananostructured surface region, a portion of which is in contact withfluid that flows through the microfluidic channel while in operation.The nanostructured surface region contains a plurality of nanostructureseach having a longitudinal dimension and a lateral dimension. Attachedto the nanostructured surface region is a plurality of binding agents,which are capable of selectively capturing target cells in a cellsample.

Embodiments of the present invention are also directed to a method ofisolating target cells from a cell sample. The method involves providinga cell sample having at least one target cell and contacting the cellsample with a plurality of nanostructures. Attached to thenanostructures is a plurality of binding agents, which is capable ofselectively capturing the target cells in the cell sample.

Other embodiments of the present invention are directed to using themethods and devices of the present invention to diagnose disease,monitor disease progression, and evaluate the efficacy of a treatment.

Further embodiments of the present invention are directed to kits thatcontain a device of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides a conceptual illustration of how a nanostructuredsubstrate can be employed to achieve improved cell-capture efficiencyfrom a sample according to an embodiment of the present invention. Morecell surface components attach onto nanostructured substrates than flatsubstrates because nanostructured substrates provide enhanced localinteraction with cell surface components.

FIGS. 2A-2C illustrate the preparation of silicon nanowires (SiNWs)according to an embodiment of the present invention. FIG. 2A showschemical etching by Ag⁺ and HF to introduce a SiNW array onto a siliconwafer. The scanning electron microscope (SEM) images reveal well-definedSiNWs with diameters ranging from 100 to 200 nm and length around 10 μm.FIG. 2B is a schematic presentation of grafting biotinylated epithelialcellular adhesion molecule antibodies (anti-EpCam) onto siliconsubstrates. FIG. 2C shows SEM images of SiNW substrates with differentSiNW lengths obtained by wet chemical etching.

FIGS. 3A-3C compare flat silicon substrates and SiNW substrates, whichare embodiments of the present invention. FIG. 3A shows fluorescencemicroscope images and SEM images of SiNW substrates and flat siliconsubstrates, on which MCF7 were captured. FIG. 3B shows fluorescencemicroscope images and SEM images of SiNW substrates and flat siliconsubstrates, on which Daubi B cells were captured. In FIGS. 3A and 3B,the SiNW substrates exhibited significantly higher cell captureefficiency than the flat ones. FIG. 3C is a schematic depiction of thephotolithography process for patterning alternating SiNW and flatsubstrates on a silicon wafer for comparing cell capture efficiency in aclose experimental setting. FIG. 3D shows an SEM image of patternedsubstrates before cell capture (top) and fluorescence images of cellscaptured on patterned substrates (bottom).

FIGS. 4A and 4B show the effects of capture times and SiNW length on thecapture efficiency of SiNW substrates, which are embodiments of thepresent invention. FIG. 4A shows the correlation between cell captureefficiency and capture times. FIG. 4B shows the correlation between cellcapture efficiency and different SiNW lengths ranging from 0 to 20 μm.

FIG. 5 compares the cell capture performance of three differentsubstrates: SiNW substrate without any surface modification (SiNW-No),SiNW substrate with streptavidin coating (SiNW-SA) and SiNW substratemodified with anti-EpCAM (SiNW-SA-EpCAM).

FIGS. 6A-6E depict a microfluidic device according to an embodiment ofthe present invention. FIG. 6A is a photograph of the microfluidic cellcapture platform. FIG. 6B is a schematic representation of theintegrated CTC capture platform composed of a capture agent-coated SiNWsubstrate and an overlaid microfluidic chaotic mixer. FIG. 6C is anoptical image of the SiNW pattern underneath chaotic mixing channels.FIG. 6D is a side-view SEM image of the well-defined SiNWs withdiameters ranging from 100 to 200 nm and lengths around 10 μm. FIG. 6Eshows how cell surface components attach onto the nanostructuredsubstrate with high efficiency, presumably because nanostructuredsubstrates enhance the local interaction with cell surface components.

FIGS. 7A-7C show the effects of flow rate on cell capture in amicrofluidic device according to an embodiment of the present invention.FIG. 7A shows the correlation between flow rate and capture yield in themicrofluidic device. FIG. 7B shows the distribution of capture cells inthe microchannel from inlet to outlet (0 to 88 cm). FIG. 7C is aphotograph of the microfluidic device.

FIG. 8 shows capture yields of PBS spiked with 100 cells per mL of threedifferent cancer cell lines: breast (MCF7), prostate (PC3), and bladder(T-24), in a microfluidic device according to an embodiment of thepresent invention.

FIG. 9 shows the capture efficiency for various target cellconcentrations, comparing whole blood to lysed blood samples, in amicrofluidic device according to an embodiment of the present invention.The plot represents number of cells spiked versus cells recovered.

FIGS. 10A-10D show the effects of capture time and SiNW length oncapture efficiency of a microfluidic device according to an embodimentof the present invention. FIG. 10A shows the correlation between cellcapture efficiency and capture times. FIG. 10B shows the correlationbetween cell capture efficiency and different SiNW lengths ranging from0 to 20 μm. FIG. 10C shows the percentage of targeted Daudi B cells thatare captured in a cell mixture at different ratios. FIG. 10D is afluorescent image of Daudi B cells captured on the SiNP substrates. Eachplot and error bar represents a mean±standard deviation from threerepeats.

FIGS. 11A-11C compare the capture capabilities of devices according toembodiments of the present invention and Cellsearch™ technology. FIG.11A shows the CTC numbers reported for 43 metastatic prostate cancerpatient samples. FIG. 11B shows the CTC numbers reported for the patientsamples using a device according to an embodiment of the presentinvention under static incubation conditions. FIG. 11C shows the CTCnumbers reported for the patient samples using a microfluidic deviceaccording to an embodiment of the present invention.

DETAILED DESCRIPTION

Some embodiments of the present invention are directed to a device thatis capable of rapidly and efficiently separating rare cells, e.g., CTCs,from biological samples. The device contains a binding agent attached toa nanostructure. Cell capture is mediated by the interaction of thetarget cell with the binding agent. In addition, the nanostructureassists in cell capture by interacting with cellular surface componentssuch as microvilli, lamellipodia, filopodia, and lipid-raft moleculargroups. In addition to accurately identifying and measuring rare cellsin biological samples, devices according to some embodiments of thepresent invention isolate rare cells that can be used in subsequentprocesses. Some embodiments of the present invention are furtherdirected to using the device in both research and clinical management,including using the device to detect, diagnosis, and monitor disease.

In some embodiments, the devices and methods of the present inventionare able to sort rare cells directly from whole blood in a single step.For example, devices and methods according to embodiments of the presentinvention are capable of utilizing whole, anticoagulated blood (althoughnot limited thereto) without any further sample treatment steps, such asdilution, centrifugation, red blood cell lysis, cell fixation, or celllabeling. This contrasts with immunomagnetic-bead-based systems, whichrequire multiple “bulk” semi-automated preparatory steps(centrifugation, washing, and incubation), resulting in loss and/ordestruction of a significant portion of the rare cells. In addition,devices and methods according to embodiments of the present inventionare capable of isolating both viable and fixed cells, whereas magneticbead-based approaches can only isolate fixed, nonviable cells.Furthermore, unlike existing microfluidic CTC-capture devices thatrequire complicated fluidic handling systems, devices according toembodiments of the present invention can achieve high cell-captureefficiency by statically incubating blood samples in the device.

Some embodiments of the devices and methods of the present invention arealso distinctive in that they use nanostructures to capture and isolatecirculating cells in a biological sample. Previous microfluidicCTC-capture devices employed microstructures to interact with targetcells. These microstructures are capable of interacting with most cells,which are usually 10-30 μm in size. However, these microstructurescannot interact with the various components on the cellular surface thatare nanoscale in size (e.g., microvilli). The nanostructures accordingto embodiments of the present invention enhance binding to the targetcell by interacting with these nanoscopic cellular surface components.

In some embodiments, the devices and methods of the present inventioncan achieve capture of rare cells at high sensitivity (e.g., percentageof patients having a tumor identified as having CTCs); high specificity(e.g., percentage of patients not having a tumor identified as nothaving CTCs); and high purity (defined as the percentage of the rarecells retained by the device relative to other cells retained by thedevice). The observed levels of sensitivity, specificity, and purity aresurprising in comparison to previous devices and methods for capturingCTCs. (See, e.g., FIG. 10).

In some embodiments, the devices and methods of the present inventionare readily adaptable for potential use in various clinical scenarios,including changes in throughput and in the binding agent, allowingcapture of any type of rare circulating cell. In addition, the devicesand methods according to some embodiments of the present invention arenot limited to identifying and isolating circulating tumor cells. Thedevices and methods according to embodiments of the present inventionare suitable for use within a range of cytological research areas. Theone-step potential and versatility of some embodiments of the presentinvention makes these embodiments conducive to point-of-care use andrapid integration into clinical practice.

Embodiments of the present invention are discussed in detail below. Indescribing embodiments, specific terminology is employed for the sake ofclarity. However, these embodiments are not intended to be limited tothe specific terminology so selected. One of ordinary skill in therelevant art will recognize that other equivalent components can beemployed and other methods developed without departing from the spiritand scope of the invention. All references cited herein are incorporatedby reference as if each had been individually incorporated.

1. DEFINITIONS

To facilitate an understanding of the present invention, a number ofterms and phrases are defined below.

As used herein, the singular forms “a”, “an”, and “the” include pluralforms unless the context clearly dictates otherwise. Thus, for example,reference to “a binding agent” includes reference to more than onebinding agent.

The term “nanostructure” refers to a structure having a lateraldimension and a longitudinal dimension, wherein the lateral dimension,the longitudinal dimension, or both the lateral and longitudinaldimensions are less than 1 mm. The shape of the nanostructure is notcritical. It can, for example, be any three dimensional surface such asa bead, particle, strand, tube, sphere, etc.

The terms “diagnostic” and “diagnosis” refer to identifying the presenceor nature of a pathologic condition and includes identifying patientswho are at risk of developing a specific disease or disorder. Diagnosticmethods differ in their sensitivity and specificity. The “sensitivity”of a diagnostic assay is the percentage of diseased individuals who testpositive (percent of “true positives”). Diseased individuals notdetected by the assay are “false negatives.” Subjects who are notdiseased and who test negative in the assay, are termed “truenegatives.” The “specificity” of a diagnostic assay is 1 minus the falsepositive rate, where the “false positive” rate is defined as theproportion of those without the disease who test positive. While aparticular diagnostic method may not provide a definitive diagnosis of acondition, it suffices if the method provides a positive indication thataids in diagnosis.

The terms “detection”, “detecting” and the like, may be used in thecontext of detecting biomarkers, or of detecting a disease or disorder(e.g., when positive assay results are obtained). In the latter context,“detecting” and “diagnosing” are considered synonymous.

The terms “subject”, “patient” or “individual” generally refer to ahuman, although the methods of the invention are not limited to humans,and should be useful in other mammals (e.g., cats, dogs, etc.).

“Sample” is used herein in its broadest sense. A sample may comprise abodily fluid including blood, serum, plasma, tears, aqueous and vitreoushumor, spinal fluid, urine, and saliva; a soluble fraction of a cell ortissue preparation, or media in which cells were grown. Means ofobtaining suitable biological samples are known to those of skill in theart.

The term “binding agent” as used herein refers to any entity orsubstance, e.g., molecule, which is associated with (e.g., immobilizedon, or attached either covalently or non-covalently to) thenanostructured surface region, or which is a portion of such surface(e.g., derivatized portion of a plastic surface), and which can undergospecific interaction or association with the target cell. A “pluralityof binding agents” can refer to a plurality of one particular bindingagent or a plurality of more than one binding agent.

An “antibody” is an immunoglobulin molecule that recognizes andspecifically binds to a target, such as a protein, polypeptide, peptide,carbohydrate, polynucleotide, lipid, etc., through at least one antigenrecognition site within the variable region of the immunoglobulinmolecule. As used herein, the term is used in the broadest sense andencompasses intact polyclonal antibodies, intact monoclonal antibodies,antibody fragments (such as Fab, Fab′, F(ab′)₂, and Fv fragments),single chain Fv (scFv) mutants, multispecific antibodies such asbispecific antibodies generated from at least two intact antibodies,hybrid antibodies, fusion proteins comprising an antibody portion, andany other modified immunoglobulin molecule comprising an antigenrecognition site so long as the antibodies exhibit the desiredbiological activity. An antibody may be of any the five major classes ofimmunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes)thereof (e.g. IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2), based on theidentity of their heavy-chain constant domains referred to as alpha,delta, epsilon, gamma, and mu, respectively. The different classes ofimmunoglobulins have different and well known subunit structures andthree-dimensional configurations. Antibodies may be naked or conjugatedto other molecules such as toxins, radioisotopes, etc.

The term “antibody fragments” refers to a portion of an intact antibody.Examples of antibody fragments include, but are not limited to, linearantibodies; single-chain antibody molecules; Fc or Fc′ peptides, Fab andFab fragments, and multispecific antibodies formed from antibodyfragments.

“Hybrid antibodies” are immunoglobulin molecules in which pairs of heavyand light chains from antibodies with different antigenic determinantregions are assembled together so that two different epitopes or twodifferent antigens may be recognized and bound by the resultingtetramer.

“Isolated” in regard to cells, refers to a cell that is removed from itsnatural environment (such as in a solid tumor) and that is isolated orseparated, and is at least about 30%, 50%, 75%, and 90% free from othercells with which it is naturally present, but which lack the markerbased on which the cells were isolated.

That a molecule (e.g., binding agent) “specifically binds” to or shows“specific binding” or “captures” or “selectively captures” a target cellmeans that the molecule reacts or associates more frequently, morerapidly, with greater duration, and/or with greater affinity with thetarget cell than with alternative substances. Thus, under designatedexperimental conditions, the specified molecule bind to the target cellat least two times the background and does not substantially bind in asignificant amount to other cells and proteins present in the sample.

“Metastasis” as used herein refers to the process by which a cancerspreads or transfers from the site of origin to other regions of thebody with the development of a similar cancerous lesion at the newlocation. A “metastatic” or “metastasizing” cell is one that losesadhesive contacts with neighboring cells and migrates via thebloodstream or lymph from the primary site of disease to invadeneighboring body structures.

2. DEVICE

A device according to an embodiment of the present invention isillustrated schematically in FIGS. 1, 2A, and 2B. The device (100 and200) contains a substrate (102 and 202) having a nanostructured surfaceregion (104 and 204). A plurality of binding agents (106 and 206) isattached to said nanostructured surface region of said substrate. Thenanostructured surface region comprises a plurality of nanostructures(such as nanostructure 108 and nanostructure 208) each having alongitudinal dimension and a lateral dimension. As a sample is placed onthe device, biological cells (110 and 210) are selectively captured bythe binding agents and the plurality of nanostructures acting incooperation.

The binding agent or agents employed will depend on the type ofbiological cell(s) being targeted. Conventional binding agents aresuitable for use in some of the embodiments of the present invention.Nonlimiting examples of binding agents include antibodies, nucleicacids, oligo- or polypeptides, cellular receptors, ligands, aptamers,biotin, avidin, coordination complexes, synthetic polymers, andcarbohydrates. In some embodiments of the present invention, bindingagents are attached to the nanostructured surface region usingconventional methods. The method employed will depend on the bindingagents and the material used to construct the device. Nonlimitingexamples of attachment methods include non-specific adsorption to thesurface, either of the binding agents or a compound to which the agentis attached or chemical binding, e.g., through self assembled monolayersor silane chemistry. In some embodiments, the nanostructured surfaceregion is coated with streptavidin and the binding agents arebiotinylated, which facilitates attachment to the nanostructured surfaceregion via interactions with the streptavidin molecules.

In some embodiments of the present invention, the nanostructuresincrease the surface area of the substrate and increase the probabilitythat a given cell will come into contact with a binding agent. In theseembodiments, the nanostructures can enhance binding of the target cellsby interacting with cellular surface components such as microvilli,lamellipodia, filopodia, and lipid-raft molecular groups. In someembodiments, the nanostructures have a longitudinal dimension that isequal to its lateral dimension, wherein both the lateral dimension andthe longitudinal dimension is less than 1 μm, i.e., nanoscale in size.In other embodiments, the nanostructures have a longitudinal dimensionthat is at least ten times greater than its lateral dimension. Infurther embodiments, the nanostructures have a longitudinal dimensionthat is at least twenty times greater, fifty times greater, or 100 timesgreater than its lateral dimension. In some embodiments, the lateraldimension is less than 1 μm. In other embodiments, the lateral dimensionis between 1-500 nm. In further embodiments, the lateral dimension isbetween 30-400 nm. In still further embodiments, the lateral dimensionis between 50-250 nm. In some embodiments, the longitudinal dimension isat least 1 μm long. In other embodiments, the longitudinal dimension isbetween 1-50 μm long. In other embodiments, the longitudinal dimensionis 1-25 μm long. In further embodiments, the longitudinal dimension is5-10 μm long. In still further embodiments, the longitudinal dimensionis at least 6 μm long.

The shape of the nanostructure is not critical. In some embodiments ofthe present invention, the nanostructure is a sphere or a bead. In otherembodiments, the nanostructure is a strand, a wire, or a tube. Infurther embodiments, a plurality of nanostructure contains one or moreof nanowires, nanofibers, nanotubes, nano-pillars, nanospheres, ornanoparticles.

The exact device geometry will be determined based on the assay. Devicesmay, or may not, include regions that allow for optical or visualinspection of the nanostructure surface.

In embodiments, high cell-capture efficiency can be achieved bystatically incubating blood samples.

An embodiment of a microfluidic device (600) according to the presentinvention is illustrated schematically in FIG. 6B. The microfluidicdevice (600) has a flow layer (602) attached to the substrate (604) toform a microfluidic channel (606). In such a device (600), a portion ofthe nanostructured surface region of the substrate (604) will be incontact with fluid that flows through the microfluidic channel. Themicrofluidic device includes at least one fluid input microchannel(608). However, the microfluidic device is not limited to only one inputmicrochannel. In some embodiments, the microfluidic device can includetwo or more input channels as well as two or more fluid sources that arein fluid connection. The microfluidic device also has one or more outputmicrochannels (610) for egress of the fluid.

Nonlimiting examples of fluids that may be introduced into a deviceinclude washing buffers, e.g., to remove nonspecifically bound cells orunused reagents, lysing reagents, or labeling reagents, e.g.,extracellular or intracellular stains. In some embodiments, devices ofthe present invention are designed to have removable covers to allowaccess to all or a region in which cells may be bound. With thesedevices, it is possible to apply reagents, e.g., labeling reagents orlysing reagents, to specific regions. Individual cells may also beremoved from such. In other embodiments, the device has more than oneinput microchannel and output microchannel to allow the introduction ofmore than one fluid to the device, typically at different times. Byhaving multiple input microchannels and corresponding outputmicrochannels, fluids may be introduced simultaneously in the device tomanipulate bound cells in specified regions. The size of these regionsmay be controlled based on the location of the input microchannels andoutput microchannels and the relative volumetric flow rates from theinput microchannels and output microchannels.

In some embodiments of the present invention, the input microchannel isconnected to a pump (612) to control the flow of sample and reagentsinto the microfluidic channel. Conventional fluid pumps capable ofproducing desired shear stress in a device are suitable for use in someembodiments of the present invention. Nonlimiting examples of pumpsinclude syringe pumps, peristaltic pumps, and vacuum sources. In someembodiments, pumps are coupled to the devices using conventionalmethods. The device may be configured for substantially constant shearstress in any given channel or variable shear stress in a given channel.One of ordinary skill in the art will know how to select and configure apump for use in the present invention based on the volume and type offluid to be processed as well as the desired fluid flow rate.

In embodiments, the device of the present invention includes a chaoticmixer. Conventional chaotic mixers are suitable for use in someembodiments of the present invention. In some embodiments, the flowlayer has a textured surface that causes chaotic flow in themicrofluidic channel. The chaotic flow increases the probability thatthe biological cells will come into contact with the nanostructuredsurface region of the substrate, thereby increasing the probability thatthe binding agents on the nanostructured surface regions will interactand bind to target biological cells in the sample. In some embodiments,the textured surface has a plurality of structures orientated relativeto a principle direction of fluid flow that mix the circulating fluid.The textured surfaces may be formed in a variety of geometrical shapes,including for example, rectangular, circular, and parabolic. The shapesmay be combined into a periodic or random arrangement. In someembodiments, the shapes may include a plurality of chevron-shapes thatform a herring-bone pattern. As used herein, the term “herring-bonepattern” has its normal meaning of columns (e.g., two) of short parallellines with all the lines in one column sloping one way and lines inadjacent column sloping the other way. Additional details about thepatterns that may be formed in the textured surfaces to facilitate fluidmixing are described in U.S. Published Patent Application 2004/0262223,titled “LAMINAR MIXING APPARATUS AND METHODS,” by Stook et al.

In some embodiments, devices of the present invention are fabricatedusing conventional techniques. The fabrication techniques employed willdepend on the material used to make the device. Nonlimiting examples offabrication techniques include molding, photolithography, electron beamlithography, soft lithography, electroforming, and machining.Nonlimiting examples of materials include glass, quartz, polymers (e.g.,polystyrene, silicones such as polydimethylsiloxane (PDMS), epoxy,polymethylmethacrylate, urethanes, polysaccharide, polylactide, andpolytetrafluoroethylene (Teflon)), silicon and other semiconductors, andmetals (e.g., aluminum, titanium, and steel). The material may also bean inorganic oxide (e.g., zinc oxide, silicon oxide, titanium oxide, andaluminum oxide).

In some embodiments of the present invention, a microfluidic device isimplemented by soft lithography. For example, a layer ofpolydimethylsiloxane (PDMS) can be applied to a substrate that has adesired pattern. The layer can be coated with resist, exposed to a lightpattern, and etched to create structures to form fluid channels, forexample, in a predefined pattern. Successive steps of coating, exposing,and etching can be used to create more complex structures.

3. METHODS OF USE

In embodiments, the devices of the present invention are employed toisolate rare cells from a sample. In some embodiments, the rare cellsare circulating tumor cells from peripheral blood. In other embodiments,the rare cells are organisms found in peripheral blood (e.g., bacteria,viruses, protists, and fungi). In further embodiments, the rare cellsare nonhemopoietic cells not normally found in blood (e.g., endothelialcells or fetal cells), and even cells of hemopoietic origin (e.g.,platelets, sickle cell red blood cells, and subpopulations ofleukocytes).

Cancers that may be detected using devices according to embodiments ofthe present invention include prostate, lung, adenocarcinoma, adenoma,adrenal cancer, basal cell carcinoma, bone cancer, brain cancer, breastcancer, bronchi cancer, cervical dysplasia, colon cancer, epidermoidcarcinoma, Ewing's sarcoma, gallbladder cancer, gallstone tumor, giantcell tumor, glioblastoma multiforma, head cancer, hyperplasia,hyperplastic corneal nerve tumor, in situ carcinoma, intestinalganglioneuroma, islet cell tumor, Kaposi's sarcoma, kidney cancer,larynx cancer, leiomyoma tumor, liver cancer, malignant carcinoid,malignant hypercalcemia, malignant melanomas, marfanoid habitus tumor,medullary carcinoma, metastatic skin carcinoma, mucosal neuromas,mycosis fungoide, neck cancer, neural tissue cancer, neuroblastoma,osteogenic sarcoma, osteosarcoma, ovarian tumor, pancreas cancer,parathyroid cancer, pheochromocytoma, primary brain tumor, rectumcancer, renal cell tumor, retinoblastoma, rhabdomyosarcoma, seminoma,skin cancer, small-cell lung tumor, soft tissue sarcoma, squamous cellcarcinoma, stomach cancer, thyroid cancer, topical skin lesion,veticulum cell sarcoma, or Wilm's tumor. In some embodiments, thebinding agents are anti-epithelial-cell adhesion molecule antibodies(anti-EpCAM antibodies). EpCAM provides specificity for CTC capture fromunfractionated blood as it is frequently overexpressed by carcinomas oflung, colorectal, breast, prostate, head and neck, and hepatic origin,and can therefore provide clinical and diagnostic information relevantto tumors, even those considered clinically localized.

In addition to methods of isolating biological cells from a sample, someembodiments of the present invention provide methods in which theisolated cells may be used to provide additional information. Inembodiments, cells isolated using the methods and devices of the presentinvention can be further assayed using additional in vitro assays. Insome embodiments, cells that are isolated using the methods and devicesof the present invention are counted. Conventional methods for countingcells can be used in some embodiments, including for example, optical,e.g., visual inspection, automated counting, microscopy based detection;FACS; and electrical detection, e.g., Coulter counters. Cell countingcan be useful for diagnosing disease, monitoring the progress ofdisease, and monitoring or determining the efficacy of a treatment.

In some embodiments, cells isolated using the methods and devices of thepresent invention are subjected to immunocytochemical analysis byflowcytometry or other analytical platforms. Such analysis facilitatesdiagnosis and provides important information to the clinician.

In some embodiments, cells isolated using the methods and devices of thepresent invention can be lysed, and one or more properties of the cells,or portions thereof, can be measured. Nonlimiting examples of biologicalproperties that can be measured in lysed cells include mRNA expression,protein expression, and DNA quantification. Additionally, in someembodiments, the cellular DNA can be sequenced, or certain sequencecharacteristics (e.g., polymorphisms and chromosomal abnormalities) canbe identified using conventional techniques, e.g., FISH or PCR. In someembodiments, cells are lysed while still bound to the device. Theability to lyse cells on the device and obtain useful geneticinformation is made possible by the high purity of samples obtainedusing devices and methods according to some embodiments of the presentinvention.

In some embodiments, cells isolated by the methods of the presentinvention are assayed without lysis. Nonlimiting examples of methods forassaying non-lysed cells include using extracellular or intracellularstains; observing morphology or growth characteristics in various media;and identifying biomarkers on the cellular surface. In furtherembodiments, the isolated cells are cultured to obtain an enrichedpopulation or the isolated cells before use in subsequent in vitroassays.

In some embodiments of the present invention, information that can beobtained from the isolated cells includes identification or enumerationof particular genomic DNA, cDNA, or mRNA sequences; identification orenumeration of cell surface markers (e.g., CD33, CD44, CD24,epithelial-specific antigen (ESA), Nanog, and BMII on cancer stemcells); and identification or enumeration of proteins or otherintracellular contents that are indicative of the type or presence of aparticular tumor. In embodiments, CTCs may be analyzed to determine thetissue of origin, the stage or severity of disease, or susceptibility toa particular treatment.

In some embodiments, the methods and devices of the present inventionare used to assess residual cancer cells in circulation followingmedical, radiation, or surgical treatment to eradicate the tumor. Infurther embodiments, the methods and devices of the present inventionare performed periodically over a course of years to assess the patientfor the presence and number of tumor cells in the circulation as anindicator of occurrence, recurrence and/or progression of disease.

Also provided in some embodiments of the present invention are kits forcarrying out the methods described herein. In embodiments, the kitcontains a device of the present invention. In some embodiments, the kitcontains reagents for use with the device of the present invention. Infurther embodiments, the kit includes instructions for taking a samplefrom a mammalian subject (e.g., body fluid), and using the kit todiagnose cancer in a mammalian subject, or monitoring the effect oftherapy administered to a mammalian subject having cancer.

Embodiments of the present invention can be further understood byreference to the following non-limiting examples. It will be apparent tothose of ordinary skill in the art that many modifications, both tomaterials and methods, may be practiced without departing from the scopeof the present disclosure.

EXAMPLES

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons of ordinary skill in theart and are to be included within the spirit and purview of thisapplication.

Example 1 Preparation and Surface Modification of SiNW Substrates

Nanostructured cell-capture substrates were prepared as follows. First,densely packed silicon nanowires (SiNWs) with diameters between 100-200nm were introduced onto silicon wafers (e.g., 1 cm×2 cm) using a wetchemical etching method (FIG. 2A). The surface of the silicon substratewas treated to become hydrophilic. The silicon wafer was sonicated inacetone and ethanol at room temperature for 10 and 5 minutes,respectively, to remove contamination from organic grease. Then, thedegreased silicon substrate was heated in boiling Piranha solution (4:1(v/v) H₂SO₄/H₂O₂) and RCA solution (1:1:5 (v/v/v) NH₃/H₂O₂/H₂O) for 1hour each, and the silicon substrate was rinsed several times withdeionized (DI) water. The clean silicon substrate was treated by a wetetching process. A Teflon vessel was used as the container, and anetching mixture consisting of DI water, HF, and silver nitrate was usedat room temperature. The concentrations of HF and silver nitrate were4.6 and 0.2 M, respectively. The etching duration was variable,depending on the required length of the nanowires. After etching, thesubstrate was immersed in boiling aqua regia (3:1 (v/v) HCl/HNO₃) for 15minutes to remove the silver film. Finally, the substrate was rinsed byDI water, dried by nitrogen, and was ready for surface modification. Thelengths of these chemically etched SiNWs can be controlled by applyingdifferent etching times. As a result, we were able to obtain a series ofSiNW substrates with SiNW lengths varying from 1 to 25 μm (FIG. 2C).After the preparation of SiNW substrates, NHS-Maleimide chemistry (FIG.2B) was employed to introduce streptavidin onto the surfaces of theSiNWs. The substrate was modified with 1% (v/v) 3-mercaptopropyltrimethoxysilane in ethanol at room temperature for 12 hours or with 4%(v/v) 3-mercaptopropyl trimethoxysilane in ethanol at room temperaturefor 45 minutes. The substrate was then treated with the coupling agentN-ymaleimidobutyryloxy succinimide ester (GMBS, 0.25 mM) for 30 min,resulting in GMBS attachment to the substrate. Next, the substrate wastreated with 10 μg/ml of streptavidin at room temperature for 30minutes, leading to immobilization onto GMBS. The substrate was flushedwith 1×PBS to remove excess streptavidin, and the streptavidin-coatedSiNW substrate was stored in 4° C. in the presence of PBS buffer (pH7.2) for up to 6 months. Biotinylated anti-EpCAM (R&D) was fleshlyintroduced onto the streptavidin-coated substrate prior to use incell-capture experiments.

Example 2 Comparison of Morphologies of Cells Captured on SiNWSubstrates and Flat Substrates

The nanoscale cell/substrate interactions were visualized using scanningelectron microscope (SEM). In order to maintain the morphologies of thesubstrate-immobilized cells, the samples were processed byglutaraldehyde fixation, osmium tetroxide treatment, and dehydration.Briefly, cells were fixed with 1.5-4% glutaraldehyde buffered in 0.1 Msodium cacodylate (4° C., 1 hr) after a 24 hour incubation onsubstrates. Cells were then post-fixed in 1% osmium tetroxide for 1 hourand 1% tannic acid was used as a mordant. Samples were dehydratedthrough a series of alcohol concentrations (30%, 50%, 70% and 90%),stained in 0.5% uranyl acetate, and followed by further dehydration(96%, 100% and 100% alcohol). The final dehydration was inhexamethyldisilazane (HMDS) followed by air drying. Once dry, thesamples were sputter coated with gold before examination with a HitachiS800 field emission SEM at an accelerating voltage of 10 keV.

The cells were also visualized using fluorescence microscopy. Controlsamples were prepared by spiking DID stained MCF7 breast cancer cellsinto rabbit blood at cell densities of 1000-1250, 80-100 and 5-20cells/mL, 25 μL of biotinylated anti-EpCAM (10 μg/mL in PBS with 1%(w/v) BSA and 0.09% (w/v) sodium azide) was added onto a 1 cm×2 cmsubstrate and incubated for 30 minutes. The substrate was washed withPBS. 1 mL of sample was added onto a substrate and incubated for 45minutes (37° C., 5% CO₂). The substrate was washed with PBS and thecells captured on the substrate were fixed with 4% paraformaldehyde(PFA) in PBS for 20 minutes. To stain and visualize captured cells, 0.9mL of 0.2% Triton X-100 in PBS was added to the substrate and incubatedfor 10 minutes. A DAPI solution (1×DAPI reagent in 1 mL of DI water) wasthen added to the substrate and incubated for 5 minutes. The substratewas washed with PBS, and the substrate was inverted onto a standardcover glass. Cells were imaged and counted using a Nikon TE2000fluorescence microscope. Color, brightness, and morphometriccharacteristics including cell size, shape, and nuclear size wereemployed to identify potential CTCs and exclude cell debris andnon-specific cells. Cells that showed dual stains (red: DiD⁺ and blue:DAPI⁺) and had certain phenotypic morphological characteristics werescored as CTCs, and DAPI⁺ cells were scored as non-specific cells.

As shown in the right of FIG. 3A, the cells captured on flat Sisubstrates show significantly different morphologies as compared tocells captured on SiNW substrates. The insets in FIG. 3A are the typicalmorphologies of cells captured on flat Si substrates (top) and SiNWsubstrates (bottom). On flat substrates, there are lamellipodia linkedlamellas surrounding the central part of cell (usually including nucleusand perinuclear organelles). These results suggest that cells begin tospread once they associate with the flat Si substrates, although thecells have difficulty attaching onto the flat Si substrates. Incontrast, a lot of filopodia protruding from cells attach onto nanowiresin three dimensions (3D) on the SiNW substrates, with either their topor middle “grasping” the nanowires. Also, the filopodia are nanoscale insize (approximately 100-150 nm) like the lateral dimension of the SiNWs.Although the SiNWs are immobile, cell surface components can arrangethemselves, resulting in more intimate local interactions between cellsand substrates. Therefore, SiNWs contribute to cell capture and accountfor the different morphologies observed with cells associated with flatSi substrates and SiNW substrates.

We also confirmed these results using Daudi B cells (i.e., cancerous Bcells) as the target cells and nanostructures coated with anti-CD20 tocapture the Daudi B cells. The results are shown in FIG. 3B and areconsistent with the results observed with MCF7 breast cancer cells.

We further compared the binding efficiency of SiNW substrates with flatSi-substrates when placed in close proximity. Photolithography was usedto apply a pattern onto the silicon substrate (the left panel in FIG.3C) in combination with a chemical etching process. We made thepatterned substrate with and without nanowires (top of FIG. 3D). After asimilar process of surface modification and cell capture as describedabove, the patterned substrate were observed under a fluorescencemicroscope. In these experiments, we used MCF7 breast cancer cells andnanostructures coated with anti-EpCAM. As shown in the bottom of FIG.3D, significantly fewer cells were observed on the flat area as comparedto the nanowire area. These results are consistent with the resultobtained above, and provide further evidence that nanowire-basedsurfaces can exhibit an amplified effect on cell capture as comparedwith flat surfaces.

Example 3 Influence of Capture Time on Cell-Capture Efficiency

To determine the minimum time required to achieve maximum cell capture,we examined cell capture performance of both of the 10 μm SiNWs and flatSi-substrates (with anti-EpCAM coating) at different incubation times.Three EpCAM-expressed cancer cells (i.e., MCF7, U87 brain cancer cellsand PC3 prostate cancer cells) were tested. FIG. 4A summarizes thecorrelation between incubation time and the number ofsubstrate-immobilized cells. In the presence of SiNW substrates, maximumcell capture was achieved at a 45 minute incubation time regardless ofthe types of cells examined. At the 45 minute time point, the 10 μmSiNWs exhibit up to 10 times better cell-capture efficiency as comparedto the flat Si-substrates. Continuous increase of cell numbers wasobserved for the flat Si-substrates; however, overall cell capturenumbers were significantly lower for flat Si-substrates than thoseobserved for the SiNW substrates.

We assessed whether this high capture yield comes from non-specificinteraction of SiNWs by performing similar cell capture experiments onthree different substrates: SiNW substrate without any surfacemodification (SiNW-No), SiNW substrate with streptavidin coating(SiNW-SA), and SiNW substrate modified with anti-EpCAM (SiNW-SA-EpCAM).The number of cells captured on SiNW-No and SiNW-SA substrates were lessthan 5% of that on the SiNW substrate (FIG. 5). Therefore, high yield ofcell capture on SiNW substrates is due to the cooperative effect ofphysical local interactions between SiNWs and cell surface componentsand chemical recognition between anti-EpCAM and EpCAM on cell surface.

Example 4 Influence of SiNW Length on Cell-Capture Efficiency

We utilized a series of SiNW substrates with SiNWs lengths of 4, 6, 8,10, and 20 μm in the cell-capture experiments. Samples containing cancercell lines (i.e., MCF7, U87 brain cancer cells, or PC3 prostate cancercells) were statically incubated on SiNW substrates and flat substrates.Anti-EpCAM was coated on both substrates, and as shown in FIG. 4B,increasing the longitudinal dimension of the SiNW resulted in increasingthe number of cells captured. When the lengths of the SiNWs were longerthan 6 μm, maximum cell capture efficiency was achieved.

Example 5 Static Capture of CTCs from Spiked Whole Blood Sample

We tested the ability of our device to perform static cell capture.Artificial CTC-containing blood samples were prepared by spikingenhanced green fluorescent protein (EGFP)-expressed U87 cells intorabbit blood with cell densities of 1000, 100 and 5 cells/mL of blood.The spiked samples were incubated on 10 μm EpCAM-coated SiNW substratesfor 45 minutes. As shown in Table 1, our approach has a high captureyield (>40%), high specificity (>40%) and high sensitivity (>90%). Theseresults indicate that the device of the present invention performssignificantly better than the current leading technology, i.e., theimmunomagnetic-bead method, which has very low sensitivity (˜20-60%) andlow specificity (˜0.1%).

TABLE 1 Range of number of CTCs/10⁹ blood cells 1000~1250 80~120 5~20Capture yield 55% 40% 65% Specificity 64% 57% 44% Sensitivity 100% 100%92%

Example 6 Preparation of the Chaotic Mixing PDMS Layer

We generated a microfluidic device of the present invention having aflow layer that produces chaotic mixing. (See FIGS. 6A and 6B). Thechaotic mixing PDMS layer was fabricated by stand soft-lithographytechnology. First, we fabricated a silicon mold with positive patterns,having a two layer SU-8 pattern. The bottom layer is the mainmicrochannel (100 um height and 2 mm width) and the top layer is theherring-bone (chaotic mixing) microchannel (25 um height). Theherring-bone structure is similar to rifling in a gun barrel, which canmake an anisotropic resistance to viscous flows. After pouring the PDMSmixture and baking it for a few hours, we get the PDMS layer with aherring-bone structure on the top of microchannels. After an inlet andoutlet is punched into the PDMS layer, the 1-3 μm thick adhesive PDMSlayer was transferred to the PDMS block through contact printing,followed by direct attachment onto the anti-EpCAM-coated SiNW substrateto give an assembled device.

Example 7 The Influence of Flow Rate on Cell-Capture Efficiency

To determine the optimized flow rate required to achieve maximum cellcapture number in the microfluidic device, we spiked breast cancer cells(i.e., MCF7) into PBS at 100 cells/ml, and captured the spiked cancercells. The microfluidic device was connected to a sample bottle.Biotinylated anti-EpCAM (10 μg/mL in PBS with 1% (w/v) BSA and 0.09%(w/v) sodium azide) was loaded into the sample bottle such that themicrofluidic device was filled with the solution. The biotinylatedanti-EpCAM solution was incubated for 30 minutes, and the microfluidicdevice was then washed with PBS. 1 mL of sample was pressured throughthe microfluidic chip at a desired flow rate, followed by washing withPBS. The microfluidic device was filled with 4% paraformaldehyde (PFA)in PBS for 20 minutes in order to fix the cells captured on thesubstrate. To stain and visualize captured cells, the PFA was replacedwith 0.2% Triton X-100 in PBS for 10 minutes followed by DAPI solution(1×DAPI reagent in 1 mL of DI water) for 5 minutes. The microfluidicdevice was washed with PBS, and the microfluidic layer was separatedfrom the substrate. The substrate was inverted onto a standard coverglass for imaging.

The calculated capture efficiency was above 90% and decreasedsignificantly at flow rates above 3 mL/hour (FIG. 7A), presumably owingto increased shear stress. The efficiency of capture was not enhanced atflow rates less than 1 mL/hour, leading us to select a flow rate of 1-2ml/hour for subsequent studies. FIG. 7A summarizes the correlationbetween flow rates and capture yields.

Example 8 Effect of EpCAM Expression Level on Different Cancer CellLines

To determine the effect of EpCAM expression on CTC capture efficiencywith a microfluidic device according to an embodiment of the presentinvention, we compared the capture yields among three cancer cell lineswith varied EpCAM expression, including breast cancer MCF-7 cells,with >500,000 antigens per cell; prostate cancer PC3 cells, withapproximately 50,000 antigens per cell; and bladder cancer T-24 cells,with approximately 2,000 antigens per cell. Each cell line was spikedinto PBS at a concentration of 100 cells/mL. Despite the varying levelsof EpCAM expression on each cell line, mean capture yield was >90% inall cases (FIG. 8). These results may be due to the amplifiedcell-substrate interactions between cells and SiNW substrates in amicrofluidic device.

Example 9 Capture CTCs from Spiked Sample with a Microfluidic Device

To test the cell capture efficiency of the microfluidic device,artificial CTC-containing blood samples were prepared by spikingDiD-stained MCF7 (breast cancer cell line) into healthy donor blood atcell densities of 5000, 1000, 500, 100 and 50 cells/mL of blood. Thespiked samples were incubated on 10 μm EpCAM-coated SiNW substrates for45 minutes. As shown in FIG. 8, our microfluidic device shows a highcapture yield (>90%), which is much higher than the immunomagnetic-beadmethod. To assess the potential spatial obstacle of red blood cells inthe flow path, these studies were repeated using lysed blood fromhealthy donors. Using whole blood and lysed samples, we obtained similarresults (FIG. 9).

We also tested the effect of capture time and SiNW length on the captureefficiency of the microfluidic device. To determine the minimum timerequired to achieve maximum cell capture, we examined cell captureperformance of both of the 10 μm SiNWs (with anti-EpCAM coating) atdifferent incubation times. Daudi B cells (i.e., cancerous B cells) andJurkat cells (i.e., cancerous T cells) were tested on anti-CD20 coatedsubstrates in the microfluidic device. FIG. 10A summarizes thecorrelation between incubation time and the number ofsubstrate-immobilized cells. In the presence of SiNW substrates, maximumcell capture was achieved at a 30 minute incubation with Daudi B cells.In contrast, cell capture was not observed with Jurkat cells lackingCD20 on the cell surface.

To assess the correlation between SiNW length and capture efficiency, weutilized a series of SiNW substrates with SiNWs lengths of 4, 6, 8, 10,and 20 μm in the cell-capture experiments. Daudi B cells and Jurkatcells were tested, and as shown in FIG. 10B, increasing the longitudinaldimension of the SiNW resulted in increasing the number of cellscaptured. Maximum cell capture efficiency was achieved when the lengthof SiNW was 6 μm.

Example 10 Comparison of Captured CTC Number Between Our MicrofluidicChip and Cellsearch™ Technology

After optimization of experimental parameters, we carried out a clinicalstudy using CTC blood samples collected from metastatic prostate cancerpatients in collaboration with the Department of Urology at UCLA underthe UCLA IRB approval (IRB #09-03-038-01). We first examined the abilityof a device according to an embodiment of the present invention tocapture CTCs under static binding conditions. Briefly, Blood sampleswere drawn from patients with advanced solid-stage tumors (as approvedby IRB) and collected into vacutainer tubes containing ETDA. 25 μL ofbiotinylated anti-EpCAM (10 μg/mL in PBS with 1% (w/v) BSA and 0.09%(w/v) sodium azide) was added onto a 1 cm×2 cm substrate and incubatedfor 30 minutes. The substrate was washed with PBS, and 1 mL of samplewas added onto the substrate and incubated for 45 minutes (37° C., 5%CO₂). The substrate was washed with PBS and the captured cells fixedwith 4% paraformaldehyde (PFA) in PBS for 20 minutes.

A 3-parameter immunocytochemistry protocol (for parallel staining ofDAPI, FITC-labeled anti-CD45 and PE-labeled anti-cytokeratin (CK)) wasapplied to stain the immobilized cells. For example, 200 μL of 0.3%Triton X-100 in PBS was added to the substrate and incubated for 30minutes. 200 μL of blocking solution (5% normal goat serum, 0.1% Tween20, 3% BSA in PBS) was added to the substrate and incubated for one hourat room temperature. Next, 200 μL of fluorophore-labeled antibodysolution (20 μL/1 mL initial concentration) was added to the substrateand incubated in the dark at 4° C. overnight. The substrate was washedwith PBS, and DAPI solution (10 μg/mL) was added and incubated for 5minutes. The substrate was washed with PBS, and the substrate wasinverted onto a standard cover glass for imaging.

We also tested the samples in a microfluidic device according to anembodiment of the present invention. The microfluidic device wasconnected to a sample bottle. Biotinylated anti-EpCAM (10 μg/mL in PBSwith 1% (w/v) BSA and 0.09% (w/v) sodium azide) was loaded into thesample bottle such that the microfluidic device was filled with thesolution. The biotinylated anti-EpCAM solution was incubated for 30minutes, and then washed with PBS. 1 of patient sample was pressuredthrough the microfluidic chip at a flow rate of 1 mL/hour. Themicrofluidic device was washed with PBS, followed by 4% paraformaldehyde(PFA) in PBS for 20 minutes in order to fix the captured cells. To stainand visualize captured cells, PFA was replaced with 0.2% Triton X-100 inPBS for 10 minutes followed by fluorophore-labeled antibody solution (20μL/1 mL initial concentration). The microfluidic device was incubated inthe dark at 4° C. overnight. The microfluidic device was then washedwith PBS, and DAPI solution (1×DAPI reagent in 1 mL of DI water) wasadded and incubated for 5 minutes. The microfluidic device was washedwith PBS, and the microfluidic layer was separated from the substrate.The substrate was inverted onto a standard cover glass for imaging

According to the signal thresholds and size/morphology featuresestablished for model cells, CTCs were clearly distinguished from thebackground immune cells. Since only 1.0 mL of patient blood is requiredfor each CTC capture study, we were able to perform 3 measurements oneach patient blood sample we received. FIGS. 11A-11C shows the resultsof CTC-capture experiments using our device under static conditions(FIG. 11B), using our device under fluid conditions (FIG. 11C), andusing the CellSearch™ technology (FIG. 11A). Our devices, under staticand fluid conditions, were able to identify CTC positive patientsamples, where the CellSearch™ technology failed to register any CTCcounts.

Example 11 Reagents

Nonlimiting examples of reagents suitable for use in practicingembodiments of the present invention include the following:

-   1. Oriented prime grade silicon wafers, p-type, resistivity of ca.    10-20 ohm-cm (Silicon Quest Int'l). Stored at room temperature.-   2. Photoresist (PR) AZ 5214 (AZ Electronic Materials USA Corp.)-   3. Developer AZ 400K (AZ Electronic Materials USA Corp.)-   4. Photoresist SU8-2100 (MicroChem. Corp. USA.)-   5. Photoresist SU8-2025 (MicroChem. Corp. USA.)-   6. Developer SU8 (MicroChem Corp, USA.)-   7. Ethanol, >99.5% (Sigma-Aldrich Co). Stored at room temperature.-   8. Sulfuric acid, 98% (Sigma-Aldrich Co, #32050-1). Stored at room    temperature.-   9. Hydrogen peroxide, 30% (Sigma-Aldrich Co, #31698-9). Stored at    room temperature.-   10. Hydrofluoric acid, 48% wt. % in H₂O (Sigma-Aldrich Co,    #339261-100 mL), Stored at room temperature.-   11. Silver nitrate, >99.8% (Sigma-Aldrich Co, #S6506-5G). Stored at    room temperature.-   12. Acetone, ACS reagent, SpectroGrade 99.5% (Fisher Scientific,    #AC40010-0040). Stored at room temperature.-   13. Isopropanol, ACS reagent, SpectroGrade 99.5% (Fisher Scientific,    # AC41279-5000). Stored at room temperature,-   14. 3-Mercaptopropyl trimethoxysilane, 95% (Sigma-Aldrich Co,    #175617-25G). Stored at room temperature.-   15. N-y-maleimidobutyryloxy succinimide ester (4-Maleimidobutyric    acid N-hydrosuccinimide, GMBS), >98% HPLC (Sigma-Aldrich Co,    #63175-25MG-F). Stored at room temperature.-   16. Streptavidin, 1 mg/mL (Invitrogen, #SNN1001). Stored in single    use aliquots at −20° C.-   17. Glutaraldehyde E.M. grade, 3% (Polysciences). Stored at room    temperature.-   18. Cacodylic acid sodium salt trihydrate (Sigma Aldrich, #C0250-10    g). Stored at room temperature.-   19, Osmium tetroxide, ACS reagent, >98% (Sigma Aldrich, #419494-250    mg). Toxic! Store at room temperature.-   20. Tannic acid (Electron Microscopy Sciences). Stored at room    temperature.-   21. Uranyl acetate (Electron Microscopy Sciences). Stored at room    temperature.-   22. Hexamethyldisilazane (HDMS) (Sigma, #H4875-100 mL). Toxic!    Stored at room temperature.-   23. Trimethylsilyl chloride (TMSCI, >98%, Alfa Aesar, #    MFCD00000502)-   24. Polydimethylsiloxane (PDMS, GE RTV 615)-   25. Breast cancer cell line, MCF7 (American Type Culture Collection)-   26. Dulbecco's Modified Eagle's Medium (DMEM, 1×), liquid (high    glucose), (Invitrogen, #11965-118)-   27. Fetal bovine serum (FBS), standard (Fisher Scientific,    #BW14-502F). Stored at −20° C.-   28. Penicillin-Streptomycin, 100× (Fisher Scientific, #ICN1670049).    Stored at −20° C.-   29. Citrated whole rabbit blood (Colorado Serum Company)-   30. Vybrant® DiD cell-labeling solution (Invitrogen, #11330-057).    Stored at 4° C.-   31. Dulbecco's phosphate buffered saline (PBS), (Invitrogen,    #14190250) Stored at 4° C.-   32. Biotinylated anti-human EpCAM/TROP1 antibody (Goat IgG, R&D)    diluted to 10 μg/mL, following the R&D product manu. Stored in    single use aliquots at −20° C.-   33. Lab-Tek chamber slides, 4 well glass, sterile (Thermo Fisher    Scientific, #177399). Stored at room temperature.-   34. Cytokeratin anti-cytokeratin PE (CAM5.2, conjugated with    phycoerythrin) (BD Biosciences, #347204) diluted to 20 μg/mL, in    PBS. Stored in single use aliquots at −20° C.-   35. FITC anti-human CD45, Ms IgG1, clone H130 (BD Biosciences,    #555482) diluted to 20 μg/mL in PBS. Stored in single use aliquots    at −20° C.-   36. 1×PBS buffer (rinsing agent). Store at 4° C.-   37. 1% DAPI in 1×PBS (nucleic staining agent). Stored at 4° C.-   38. 4% paraformaldehyde in 1×PBS (fixing agent). Stored at 4° C.-   39. Bovine serum albumin (BSA), (Sigma). Stored at 4″C.-   40. Triton X-100. Stored at 4° C.

Example 12 Methods for Practicing Embodiments of the Present Invention

Nonlimiting examples of methods for making and practicing embodiments ofthe present invention include the following:

-   1. Cut silicon wafer into pieces of silicon substrates having an    area of 1 cm×2 cm.-   2. Ultrasonicate the cut silicon substrate in acetone for 10 min. at    room temperature and dry it under nitrogen gas. Next, ultrasonicate    substrate in ethanol for five minutes at room temperature and dry    under nitrogen gas. These steps remove contamination (such v/as    organic grease) from the silicon substrate.-   3. To etch the silicon wafer surface, first heat the silicon    substrate in boiling Piranha solution (4:1 (v/v) H₂SO₄/H₂O₂) for 1    hr. Then, heat substrate in boiling RCA solution (1:1:5 (v/v)    NH₃/H₂O₂/H₂O) for 1 hr. Rinse the substrate five times with DI    water. Next, place the silicon substrates in a Teflon vessel and    etch substrate with etching mixture (4.6 M HF, 0.2 M silver nitrate    in DI water) at 50° C.-   4. Immerse substrate in boiling aqua regia (3:1 (v/v) HCl/HNO₃) for    15 min.-   5. Rinse substrate with DI water and dry under nitrogen gas.-   1. Add 4% (v/v) 3-mercaptopropyl trimethylsilane in ethanol to the    substrate, then leave the substrate in room temperature for 45 min.-   2. Treat substrate with 0.25 mM N-maleimidobutyryloxy succinimide    ester (GMBS) and incubate for 30 min at room temperature.-   3. Treat substrate with 10 μg/mL streptavidin (SA) and incubate for    30 minutes at room temperature.-   4. Flush substrate with 1×PBS to remove excess streptavidin.-   5. Store modified substrates at 4-8° C. for up to 6 months.-   1. Allow cells to incubate on the substrates for 24 hours.-   2. Fix cells with 4% glutaraldehyde buffered in 0.1 M sodium    cacodylate and incubate cells for 1 hr at 4° C. Afterward, fix cells    using 1% osmium tetroxide for 1 hr. Use 1% tannic acid as a mordant.-   3. Dehydrate samples through a series of alcohol concentrations    (30%, 50%, 70%, and 90%). After dehydrating, stain samples with 0.5%    uranyl acetate. Further dehydrate the samples through a series of    higher alcohol concentrations (96%, 100%, and 100%). Dehydrate    samples for the final time in hexamethyldisilazane (HMDS).-   4. Air dry samples.-   5. Once dry, sputter coat samples with gold and examine with a field    emission SEM (accelerating voltage of 10 keV).-   1. Stain MCF7 cells with DiD red fluorescent dye.-   2. Prepare control samples by spiking stained MCF7 cells into rabbit    blood with cell densities of 1000-1250, 80-100 and 5-20 cells/mL.-   1. Place substrates into a size-matched 4-well Lab-Tek Chamber    Slide. Drop 25 μL of biotinylated anti-EpCAM (10 μg/mL in PBS with    1% (w/v) BSA and 0.09% (w/v) sodium azide) onto a 1 cm×2 cm    substrate. Incubate for 30 min. Wash with PBS.-   2. Load 1 mL of control sample onto each substrate.-   3. Incubate device setup for 45 min (37° C., 5% CO₂).-   4. Gently wash substrate with PBS at least 5 times.-   5. Fix cells captured on substrate with 4% paraformaldehyde (PFA) in    PBS for 20 min.-   6. To stain and visualize captured cells, treat substrate with 0.9    mL of 0.2% Triton X-100 in PBS and incubate for 10 min. Incubate    substrate with a DAPI solution (1×DAPI reagent in 1 mL of DI water)    for 5 min. Wash the substrate three times with PBS. Invert substrate    onto a standard cover glass.-   7. Image and count cells using a Nikon TE2000 fluorescence    microscope.-   8. Cells that show dual stains (red: DiD+ and blue: DAPI+) and meet    the phenotypic morphological characteristics are scored as CTCs.    Color, brightness, and morphometric characteristics including cell    size, shape, and nuclear size are employed to identify potential    CTCs and exclude cell debris and non-specific cells. Cell counts by    DAPI+ only are non-specific cells.-   9. These procedures should maximize the efficiency of blood sample    preparation.-   1. Place substrates into a size-matched 4-well Lab-Tek Chamber    Slide. Drop 25 μL of biotinylated anti-EpCAM (10 μg/mL in PBS with    1% (w/v) BSA and 0.09% (w/v) sodium azide) onto a 1 cm×2 cm    substrate. Incubate for 30 min. Wash with PBS.-   2. Blood samples drawn from patients with advanced solid-stage    tumors (as approved by IRB) are collected into vacutainer tube    containing the anticoagulant ETDA. Samples should be processed    immediately after collection.-   3. To capture cells, load 1 mL of patient sample onto each SiNP    substrate. Incubate device setup for 45 min (37° C., 5% CO₂). Gently    wash the substrate with PBS at least 5 times.-   4. Fix cells captured on substrate by loading 200 μL of 4%    paraformaldehyde (PFA) in PBS onto each substrate for 20 min at room    temperature. Wash each substrate three times with PBS.-   5. Permeabilize cells by treating each substrate with 200 μL of 0.3%    Triton X-100 in PBS for 30 min at room temperature. Subsequently,    wash each substrate three times with PBS.-   6. Add 200 μL of blocking solution (5% normal goat serum, 0.1% Tween    20, 3% BSA in PBS) to each substrate and incubate for one hour at    room temperature. Next, add 200 μL of fluorophore-labeled antibody    solution (20 μL/1 mL initial concentration) to each substrate and    incubate the substrate in the dark at 4° C. overnight. Wash with 200    μL PBS three times (First wash for 15 min at room temperature,    second and third washes for 5 minutes at room temperature). Incubate    substrate with DAPI solution (10 μg/mL) for 5 minutes. Wash each    substrate 3 times with PBS.-   7. Gently invert substrate, using tweezers, onto a cover glass to    prepare for imaging.-   1. Select fluorescent microscope settings.    -   1.1. Optimized exposure times        -   1.1.1. DAPI (blue) filter: 50 ms exposure time (background:            ˜1300)        -   1.1.2. FITC (green) filter: 300 ms exposure time            (background: ˜1600)        -   1.1.3. TRITC (red) filter: 100 ms exposure time (background:            ˜1300)        -   1.2. Digitizer should be set to 1 MHz. Other settings will            yield very high background in green filter.-   2. Place sample on microscope and focus on edge of substrate. Once    focused, switch filter to DAPI    -   2.1. Starting in the upper right corner of the substrate, scan        for nuclei that are approx. 7 to 20 μm in diameter at 4× or 10×        magnification.    -   2.2. Increase magnification to 10× or 20× when a putative cell        has been located. Using a microscope mouse, check fluorescence        intensity under DAPI, TRITC, and FITC fluorescence. Sample        fluorescence intensity >2× background fluorescence intensity is        scored as a positive result.-   3. Cells that demonstrate dual staining (red: anti-cytokeratin PE⁺    and blue: DAPI⁺) and meet standard phenotypic and morphological    characteristics should be scored as CTCs. Cells that show dual    staining (green: anti-CD45 FITC⁺ and blue: DAPI⁺) should be excluded    as lymphocytes/non-specific cells. Items that demonstrate staining    in all three filters (green+ and red+ and blue+) should be excluded    as cell debris.-   1. Cleaning wafer: acetone, ethanol & DI water.-   2. Drying wafer: blow dry wafers & put on hot plate 150° C. for 5    min.-   3. Pour AZ 5214 onto wafer.-   4. Spin the wafer at 1000 rpm for 1 min.-   5. Soft bake wafers at 100° C. for 1 min.-   6. Expose wafers for UV for 56 mJ/cm².-   7. Prepare developing solution: AZ 400K: water=1:3.-   8. Develop AZ 5214 with the developing solution.-   9. Blow dry the product.-   10. Hard bake AZ 5214 at 100° C. for 5 min.-   11. To etch the AZ 5214 patterned silicon wafer surface, place the    silicon substrates in a Teflon vessel and etch substrate with    etching mixture (4.6 M HF, 0.2 M silver nitrate in DI water) at 50°    C.-   12. Immerse substrate in boiling aqua regia (3:1 (v/v) HCl/HNO₃) for    15 min.-   13. Rinse substrate with DI water and dry under nitrogen gas.-   1. Cleaning wafer: acetone, ethanol & DI water.-   2. Drying wafer: blow dry wafers & put on hot plate 150° C. for 5    min.-   3. Pour SU8-2100 onto wafer.-   4. Spin the wafer at 3000 rpm for 1 min.-   5. Soft bake wafers at 95° C. for 15 min.-   6. Expose wafers for UV for 320 mJ/cm².-   7. Post bake wafers at 95° C. for 10 min-   8. Develop SU8-2100 with the SU-8 developing solution.-   9. Blow dry the product.-   10. Hard bake SU8-2100 at 150° C. for 5 min.-   11. Pour SU8-20250 onto wafer.-   12. Spin the wafer at 2000 rpm for 1 min.-   13. Soft bake wafers at 95° C. for 8 min.-   14. Expose wafers for UV for 250 mJ/cm².-   15. Post bake wafers at 95° C. for 5 min-   16. Develop SU8-2025 with the SU-8 developing solution.-   17. Blow dry the product.-   18. Hard bake SU8-2025 at 150° C. for 10 min.-   1. Expose the silicon mold for microfluidic chip to TMSCI vapor for    10 min.-   2. Pour well mixed PDMS pre-polymer (GE RTV 615 A and B, total 20 g,    mixing ratio A:B=10:1) onto the silicon mold.-   3. Bake the pre-polymer at 80° C. for 48 h.-   4. Peel PDMS layer off from the silicon mold and punch holes for    inlet and outlet.-   5. Clap the PDMS layer onto the Streptavidin-Coated SiNP Substrates    to form a microfluidic chip,-   1. Stain MCF7 cells with DiD red fluorescent dye.-   2. Prepare control samples by spiking stained MCF7 cells into rabbit    blood with cell densities of 1000-1250, 80-100 and 5-20 cells/mL.-   1. Connect the microfluidic chip to the sample bottle. Load 25 μL of    biotinylated anti-EpCAM (10 μg/mL in PBS with 1% (w/v) BSA and 0.09%    (w/v) sodium azide) into the sample bottle. Fill full of the    microfluidic chip with the solution. Incubate for 30 min. Wash with    PBS.-   2. Pressure 1 mL of artificial blood sample through the microfluidic    chip at the desired flow rate.-   3. Gently wash microfluidic chip with 100 μL PBS.-   4. Fill full of the microfluidic chip with 4% paraformaldehyde (PFA)    in PBS for fixing cells captured on substrate for 20 min,-   5. To stain and visualize captured cells, replace the PFA with 0.2%    Triton X-100 in PBS and incubate for 10 min.-   6. Replace the Triton X-100 with a DAPI solution (1×DAPI reagent in    1 mL of DI water) for 5 min.-   7. Wash the microfluidic chip with 200 μL PBS.-   8. Unclap the microfluidic layer from substrate and invert substrate    onto a standard cover glass.-   9. Image and count cells using a Nikon TE2000 fluorescence    microscope.-   10. Cells that show dual stains (red: DiD+ and blue: DAPI+) and meet    the phenotypic morphological characteristics are scored as CTCs.    Color, brightness, and morphometric characteristics including cell    size, shape, and nuclear size are employed to identify potential    CTCs and exclude cell debris and non-specific cells. Cell counts by    DAPI+ only are non-specific cells.-   1. Connect the microfluidic chip to the sample bottle. Load 100 μL    of biotinylated anti-EpCAM (10 μg/mL in PBS with 1% (w/v) BSA and    0.09% (w/v) sodium azide) into the sample bottle. Fill full of the    microfluidic chip with the solution. Incubate for 30 min. Wash with    PBS.-   2. Pressure 1 mL of patient blood sample through the microfluidic    chip at the flow rate (1 mL/h).-   3. Gently wash microfluidic chip with 100 μL PBS.-   4. Fill full of the microfluidic chip with 4% paraformaldehyde (PFA)    in PBS for fixing cells captured on substrate for 20 min.-   5. To stain and visualize captured cells, replace the PFA with 0.2%    Triton X-100 in PBS and incubate for 10 min.-   6. Next, fill the microfluidic chip with 100 μl of    fluorophore-labeled antibody solution (20 μL/1 mL initial    concentration) and incubate the microfluidic chip in the dark at    4° C. overnight. Wash the microfluidic chip with 200 μL PBS at flow    rate 1 mL/h.-   7. Fill 100 μL DAPI solution (1×DAPI reagent in 1 mL of DI water)    into microfluidic chip and incubate for 5 min. Wash the microfluidic    chip with 200 μl. PBS.-   8. Unclap) the microfluidic layer from substrate and invert    substrate onto a standard cover glass.-   1. Select fluorescent microscope settings.    -   1.1. Optimized Exposure Times        -   1.1.1. DAPI (blue) filter: 50 ms exposure time (background:            ˜1300)        -   1.1.2. FITC (green) filter: 300 ms exposure time            (background: ˜1600)        -   1.1.3. TRITC (red) filter: 100 ms exposure time (background:            ˜1300)    -   1.2 Digitizer should be set to 1 MHz. Other settings will yield        very high background in green filter.-   2. Place sample on microscope and focus on edge of substrate. Once    focused, switch filter to DAPI    -   2.1. Starting in the upper right corner of the substrate, scan        for nuclei that are approx. 7 to 20 μm in diameter at 4× or 10×        magnification.    -   2.2. Increase magnification to 10× or 20× when a putative cell        has been located. Using a microscope mouse, check fluorescence        intensity under DAPI, TRITC, and FITC fluorescence. Sample        fluorescence intensity >2× background fluorescence intensity is        scored as a positive result.-   3. Cells that demonstrate dual staining (red: anti-cytokeratin PE+    and blue: DAPI+) and meet standard phenotypic and morphological    characteristics should be scored as CFCs. Cells that show dual    staining (green: anti-CD45 FITC+ and blue: DAPI+) should be excluded    as lymphocytes/non-specific cells. Items that demonstrate staining    in all three filters (green+ and red+ and blue+) should be excluded    as cell debris.

All publications cited herein are hereby incorporated by reference intheir entirety.

REFERENCES CITED HEREIN ARE LISTED BELOW FOR CONVENIENCE

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We claim:
 1. A microfluidic device for capturing biological cells,comprising: a substrate comprising a nanostructured surface region; aplurality of binding agents attached to said nanostructured surfaceregion of said substrate; and a flow layer attached to said substrate toform a microfluidic channel such that at least a portion of saidnanostructured surface region of said substrate is in contact with fluidthat flows through said microfluidic channel while in operation, whereinsaid nanostructured surface region comprises a plurality ofnanostructures each having a longitudinal dimension and a lateraldimension, said longitudinal dimension being at least ten times greaterthan said lateral dimension, wherein biological cells are selectivelycaptured by said binding agents and said plurality of nanostructuresacting in cooperation, and wherein said flow layer comprises a texturedsurface that causes chaotic flow to increase the probability that saidbiological cells come into contact with said nanostructured surfaceregion of said substrate to be captured.
 2. A microfluidic deviceaccording to claim 1, wherein said substrate is a silicon substrate andsaid plurality of nanostructures are silicon nanowires or nanofibers. 3.A microfluidic device for capturing biological cells according to claim2, wherein said longitudinal dimension of said nanostructures is atleast 1 μm long, and said lateral dimension of said nanostructures isgreater than 30 nm and less than 400 nm, wherein said nanostructuredsurface region of said substrate is coated with streptavidin and saidplurality of binding agents are biotinylated and attached to saidnanostructured surface region of said substrate coated withstreptavidin, and wherein said plurality of binding agents is aplurality of anti-epithelial-cell adhesion molecule antibodies(anti-EpCAM).
 4. A microfluidic device according to claim 1, whereinsaid substrate is an inorganic oxide substrate comprising at least oneof zinc oxide, silicon oxide, titanium oxide or aluminum oxide and saidplurality of nanostructures are inorganic oxide nanowires or nanofibers.5. A microfluidic device according to claim 1, wherein said substrate isa polymer substrate comprising at least one of polymethylmethacrylate,polysaccharide, or polylactide and said plurality of nanostructures arepolymer nanowires or nanofibers.
 6. A microfluidic device according toclaim 1, wherein said longitudinal dimension is at least 1 μm long, andsaid lateral dimension is greater than 30 nm and less than 400 nm.
 7. Amicrofluidic device according to claim 1, wherein said nanostructuredsurface region of said substrate is coated with streptavidin and saidbinding agents are biotinylated and attached to said nanostructuredsurface region of said substrate coated with streptavidin.
 8. Amicrofluidic device according to claim 1, wherein said plurality ofbinding agents comprises one or more of DNA, peptides, aptamers, andantibodies.
 9. A microfluidic device according to claim 1, wherein saidplurality of binding agents is a plurality of anti-epithelial-celladhesion molecule antibodies (anti-EpCAM).
 10. A microfluidic device forcapturing biological cells, comprising: a substrate comprising ananostructured surface region; a flow layer attached to said substrateto form a microfluidic channel such that at least a portion of saidnanostructured surface region of said substrate is in contact with fluidthat flows through said microfluidic channel while in operation; and aplurality of binding agents attached to said nanostructured surfaceregion of said substrate, wherein said nanostructured surface regioncomprises a plurality of nanostructures each having a longitudinaldimension and a lateral dimension, wherein biological cells areselectively captured by said binding agents and said plurality ofnanostructures acting in cooperation, and wherein said flow layercomprises a textured surface that causes chaotic flow to increase theprobability that said biological cells come into contact with saidnanostructured surface region of said substrate to be captured.
 11. Amicrofluidic device according to claim 10, wherein said substrate is asilicon substrate and said plurality of nanostructures are siliconnanowires or nanofibers.
 12. A microfluidic device according to claim10, wherein said substrate is an inorganic oxide substrate comprising atleast one of zinc oxide, silicon oxide, titanium oxide or aluminum oxideand said plurality of nanostructures are inorganic oxide nanowires ornanofibers.
 13. A microfluidic device according to claim 10, whereinsaid substrate is a polymer substrate comprising at least one ofpolymethylmethacrylate, polysaccharide, or polylactide and saidplurality of nanostructures are polymer nanowires or nanofibers.
 14. Amicrofluidic device according to claim 10, wherein said longitudinaldimension is at least 1 μm long, and said lateral dimension is greaterthan 30 nm and less than 400 nm.
 15. A microfluidic device according toclaim 10, wherein said nanostructured surface region of said substrateis coated with streptavidin and said binding agents are biotinylated andattached to said nanostructured surface region of said substrate coatedwith streptavidin.
 16. A microfluidic device according to claim 10,wherein said plurality of binding agents comprises one or more of DNA,peptides, aptamers, and antibodies.
 17. A microfluidic device accordingto claim 10, wherein said plurality of binding agents is a plurality ofanti-epithelial-cell adhesion molecule antibodies (anti-EpCAM).
 18. Amicrofluidic device according to claim 10, wherein said textured surfacecomprises a plurality of structures orientated relative to a principledirection of fluid flow.
 19. A microfluidic device according to claim18, wherein said plurality of structures is oriented in a herring-bonepattern.